ChemBioChem

L-DOPA is a key therapeutic agent for Parkinsons disease, with growing demand due to global population aging. Here we report that heme-dependent tyrosine hydroxylase (TyrH) can utilize an ascorbate/O2 system--as an alternative to H2O2--to synthesize L-DOPA with markedly enhanced operational stability. While exogenous H2O2 rapidly inactivates TyrH within minutes, sodium ascorbate (NaAsc) enables sustained catalysis for up to 24 h, surpassing the H2O2-driven yield after only 30 min. UV-vis spectroscopy confirms that H2O2 readily degrades the heme center, whereas the heme remains intact in the presence of NaAsc. QM/MM simulations reveal that in situ generated H2O2 leads to the active species of Compound I for tyrosine hydroxylation. Through systematic optimization, we establish efficient reaction conditions (40 {micro}M TyrH, 1 mM L-Tyr, 100 mM NaAsc, pH 8.5, 40 {degrees}C), achieving >95% conversion of L-Tyr to L-DOPA within 2 h. This work not only provides a robust and sustainable biocatalytic route for L-DOPA production but also highlights the broader applicability of the ascorbate/O2 pathway in heme-enzyme catalysis.

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1e56ffborg.highwire.dtl.DTLVardef@1ac8b5org.highwire.dtl.DTLVardef@6f23c1org.highwire.dtl.DTLVardef@945c54_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

Intrinsically disordered enzymes serve as useful models to understand their catalytic function against the backdrop of an unstructured protein. The characteristic flexibility in conformation seen in IDPs is a rare occurrence among enzymes and one such enzyme is the engineered protein: monomeric Chorismate Mutase (mCM). mCM surprisingly retains similar enzyme activity as its parent dimeric protein Chorismate Mutase from Methanococcus jannaschii (MjCM) despite losing the ordered globular structure. In this work using a previously demonstrated transition state analogue (TSA), we analyze the structural transitions in mCM during catalysis. Additionally, consequences of three non-active site single point mutations were investigated using CD; Trp-Dansyl FRET measurements using fluorescence lifetime; and time-resolved fluorescence anisotropy measurements; to map the local (near Trp) and global structural transitions in mCM during catalysis. Mutant2 (W24K + C69A); and Mutant3 (W24K + C69A + A6C); revealed a 97 and 89% drop-in activity compared to mCM; quite unlike Mutant1 (W24K, 19% drop). Mutant1 as opposed to Mutant3 was most sensitive to binding of TSA as quantified by structural displacement measured using FRET. This was consistent with an overall globular structure compaction induced by TSA binding in Mutant1 as reflected by a dip in rotational correlation time of Cys-conjugated dansyl probe from 10.3 to 8.4 ns. Our results highlight the critical role of Cys69 residue, that is ~19 [A] away from mCM active site, in influencing the hydrophobic collapse upon substrate binding and subsequent catalytic activity.

Bacterial microcompartments (BMCs) are a diverse and widespread class of protein-based organelle consisting of a semi-permeable protein shell encapsulating an enzymatic core. Along with their native assembly pathway, isolated BMC shell proteins have been shown to assemble into alternative superstructures such as flat sheets and nanotubes. The self-assembly and modularity of BMC shell proteins make them of great interest as modular platforms for applications involving scaffolding, immobilization and compartmentalization. While the assembly of BMC shell proteins into higher-order structures has been well-studied, the design of controllable and modular cargo loading is underdeveloped in comparison. Recently, we reported the pH-controlled assembly of CcmK2 - the major hexameric shell protein of the {beta}-carboxysome BMC - into monodisperse mesh-like microscale particles. Here, we develop a suite of encapsulation strategies for stochastic or targeted loading of various cargos, as well as the direct conjugation of cargo to CcmK2 particles. Our systematic analysis demonstrates that cargo loading and particle assembly can be modulated by the choice of recruitment strategy and the order of cargo introduction. Our findings also reveal a cooperative cargo loading mechanism during assembly that influences particle sizing and apparent morphology. Our study serves as a blueprint for the rational design of tunable cargo loading into engineered BMC-derived microcompartment systems for diverse biotechnological applications.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of Nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

Cell-free expression systems (CFES) are increasingly used alongside conventional biotechnological approaches to accelerate early-stage prototyping and are particularly valuable in point-of-use settings. However, their broader adoption remains limited by time- and cost-intensive preparation, as well as stringent cryogenic storage requirements. To address this, several studies have explored lyophilization with protective additives to generate stable, solid-state CFES. These approaches had to balance the protection gained with a loss of activity due to the additives. In this study, we present a CFES that contains a tardigrade-derived Cytosolic-Abundant Heat-Soluble (CAHS) protein to protect the biosynthetic machinery in lysates from damages during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity during low-cost room temperature desiccation, while unprotected lysates are affected in mRNA synthesis kinetics and translation yields. The diversity of tardigrade-derived protective proteins is a treasure trove for cell-free synthetic biology, in particular for making CFES more accessible and portable. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/715078v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@8ecc2eorg.highwire.dtl.DTLVardef@ff0432org.highwire.dtl.DTLVardef@6c940eorg.highwire.dtl.DTLVardef@6c5390_HPS_FORMAT_FIGEXP M_FIG C_FIG

Solid-phase peptide synthesis (SPPS) remains the dominant technique for peptide production. However, its reliance on hazardous organic solvents such as N, N-dimethylformamide (DMF) and dichloromethane (DCM) results in an adverse environmental burden. One potential approach is replacing these organic solvents with water to reduce the hazardous solvent consumption and improve the environmental footprint of peptide production. This has led to the emergence of aqueous solid-phase peptide synthesis (ASPPS) approaches. Although successful, these approaches require specialized hydrophilic resins or modified building blocks, limiting their industrial applicability and scalability. Moreover, conventional hydrophobic polystyrene supports, remain the most widely used solid supports in industrial SPPS due to their high loading capacity, mechanical robustness, and low cost. These resins are generally considered incompatible with aqueous conditions. Here, we demonstrate that industrially relevant 2-chlorotrityl chloride (CTC) polystyrene resin can support efficient peptide coupling under fully aqueous conditions by integrating a precipitate-free 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC{middle dot}HCl) and Oxyma activation system with a synergistic thermal-acoustic strategy. We posit that heating combined with ultrasonic irradiation likely promotes transient relaxation of the polystyrene matrix and enhances water penetration. This facilitates the diffusion of activated amino acid esters onto the hydrophobic resin required for coupling. The robustness of this aqueous methodology was validated through the synthesis of nine structurally diverse peptide sequences, including aromatic hydrogel-forming peptides, opioid peptides derived from enkephalins, toxin-inspired sequences, and a lipid-interacting fragment of -synuclein. Analytical characterization by HPLC and MALDI-TOF mass spectrometry confirmed successful peptide assembly with high crude purity. We anticipate that this thermal-acoustic aqueous SPPS strategy provides a scalable and accessible pathway toward sustainable peptide manufacturing on classical hydrophobic supports with aqueous chemistry.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ternary composite systems formed by lactoferrin (LF), sodium alginate (Alg), and Fe(II) were designed to investigate their potential as an iron delivery platform with enhanced protein stability. The ternary LF-Alg-Fe (LAF) composites demonstrated distinct structures depending on the LF to Alg ratio and the Fe(II) concentrations. At an LF to Alg ratio of 8:2 and final Fe concentrations between 20-30 mM, the system formed complexes stabilized by electrostatic interactions. Whereas Alg-rich formulations formed hydrogels stabilized by Alg-Fe(II) egg-box cross-linking. Rheological analysis and swelling behavior indicated a higher mechanical strength in LF-rich complexes and stronger network integrity in Alg-rich hydrogels, while intermediate LF/Alg ratios showed weaker structures overall. Fourier-transform infrared spectroscopy (FTIR) spectra showed no changes in functional groups or polymer structures after composite formation, confirming composite formation via non-covalent interactions. Thermal studies indicated that these ternary systems improved LF stability, evidenced by preserved secondary structure after heating using circular dichroism (CD), and an increased denaturation temperature compared with free LF in differential scanning calorimetry (DSC). In addition, in LF-rich formulations the Fe(II) release in aqueous solution was [~]50% while in Alg-rich formulations it was much lower (< 10%). LF-Alg-Fe composites exhibit distinct structures governed by protein-polysaccharide interactions and iron-mediated cross-linking, providing a potential strategy for protein stabilization and iron fortification in food systems.

The microbial extracellular matrix (ECM) is a complex network of self-secreted biopolymers uniting the cells in biofilms, providing them with structural integrity, and contributing to their elevated resistance to antibiotic treatments. Recently, there is a growing realization that a regulated, bidirectional cross-talk of bacteria and ECM confers biofilms with tissue-like traits, however, the mechanisms of spatio-temporal self-organisation of ECM and its regulation are still poorly understood. In the model organism for biofilm formation Bacillus subtilis, TasA is the major protein component of the extracellular matrix. We recently showed that TasA, isolated in the form of stable and structured globules, assembles into elongated and ordered fibers via a donor-strand complementation mechanism. In this study, we discovered that in the presence of zinc metal ions, TasA is able to form hydrogels with > 97% water content. Electron- and atomic force-microscopies as well as small angle X-ray scattering measurements show that cross-linking with zinc ions induces a transition in TasA morphology from one-dimensional fibers to two-dimensional sheets. Electron paramagnetic resonance measurements then show that such a significant morphological shift is associated with molecular changes in the coordination environment of zinc ions, which lead to structural changes at the protein level. When assembling into macroscopic networks, TasA-Zn metallogels exhibit viscoelastic properties and a fast recovery following an excessive strain. These metallogels represent a novel class of bacterially-derived ECMs that form easily at room temperature without covalent crosslinking, and may be used as a natural matrix-mimics in biofilm models for infection studies.

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

KRAS mutations drive multiple cancers and represent an important therapeutic target, together with other oncogenic regulators such as MYC, KIT, and BCL2 that are critically involved in pancreatic cancer. Here we describe a novel therapeutic strategy based on stable nucleolipid-modified G-quadruplexes (NLG4). Cell viability assays demonstrate that NLG4 strongly inhibit pancreatic cancer cell proliferation, whereas non-lipidic G-quadruplex sequences display minimal activity under comparable conditions. Owing to their distinctive physicochemical properties, including stabilization of parallel G-quadruplex structures and self-assembly into micellar aggregates, NLG4 efficiently internalize into cells and interact with key G-quadruplex unfolding factors such as UP1. This interaction leads to a marked downregulation of KRAS, c-MYC, c-KIT, and BCL2 expression. Suppression of these oncogenes profoundly affects pancreatic cancer cell fate, as evidenced by reduced expression of proliferation (Ki67) and anti-apoptotic (BCL2) markers. In addition, NLG4 treatment decreases inflammatory signaling mediated by NF-{kappa}B and inhibits major pro-proliferative kinase pathways, including ERK, AKT, and phosphorylated AKT. The therapeutic relevance of this decoy strategy is further supported by the observed potentiation of gemcitabine antitumor activity. Overall, these findings highlight NLG4 as a promising anticancer approach that simultaneously targets multiple oncogenic pathways through G-quadruplex-based decoy mechanisms, with translational potential for future pancreatic cancer treatment.

Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum, and several fungal species of the genus Aspergillus, including Aspergillus terreus and Aspergillus fumigatus.. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus (AfKdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed AfKdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H2, making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O2 sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani, has exhibited exceptional O2-stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O2-induced degradation by formation of the Hinact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe]H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the Hinact and Htrans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydAPDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fed center of [2Fe]H, thereby reducing Hinact accumulation. Moreover, as-isolated ToHydAPDT predominantly exhibits the Hhyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe]H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.